Reconstitution of microtubule into GTP-responsive nanocapsules

Nanocapsules that collapse in response to guanosine triphosphate (GTP) have the potential as drug carriers for efficiently curing diseases caused by cancer and RNA viruses because GTP is present at high levels in such diseased cells and tissues. However, known GTP-responsive carriers also respond to adenosine triphosphate (ATP), which is abundant in normal cells as well. Here, we report the elaborate reconstitution of microtubule into a nanocapsule that selectively responds to GTP. When the tubulin monomer from microtubule is incubated at 37 °C with a mixture of GTP (17 mol%) and nonhydrolysable GTP* (83 mol%), a tubulin nanosheet forms. Upon addition of photoreactive molecular glue to the resulting dispersion, the nanosheet is transformed into a nanocapsule. Cell death results when a doxorubicin-containing nanocapsule, after photochemically crosslinked for properly stabilizing its shell, is taken up into cancer cells that overexpress GTP.


1-2. General procedures
1 H NMR and 13 C NMR spectra were recorded on a JEOL model GSX-500 spectrometer, where chemical shifts (δ in ppm) were determined using CHCl3 (δ 7.26), CHD2(CD3)SO (δ 2.50), and HDO (δ 4.79) for 1 H NMR and CDCl3 (δ 77.2) and (CD3) 2SO (δ 39.5) for 13 C NMR as internal S4 references. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was performed with a-cyano-4-hydroxycinnamic acid (CCA), 2,5-dihydroxybenzoic acid (DHB), or sinapinic acid (SA) as a matrix and an Applied Biosystems Biospectrometry Analyses of images of the acrylamide gels were performed using an Amersham Biosciences model Typhoon 9410 variable image analyser. Recycling preparative gel permeation chromatography (GPC) was performed with a Japan Analytical Industry model LC908-C60 using a column set consisting of JAIGEL 1H-40 and 2H-40. Transmission electron microscopy (TEM) images were recorded using a JEOL model JEM-1400 electron microscope operating at an anode voltage of 120 kV. Samples were applied to an electron microscope specimen grid covered with a thin carbon support film that had been hydrophilized by ion bombardment. Then, the samples were negatively stained with a saturated uranyl acetate solution. Tapping-mode atomic force microscopy (AFM) of air-dried samples on a mica surface was performed using an SII Nano Technology model NanoNavi S-image. Small-angle X-ray scattering (SAXS) was carried out at BL45XU in SPring-8 (Hyogo, Japan) S1 with a Dectris model Pilatus 3X 300K-W detector. Scattering vector q (q = 4πsinθ/λ; 2θ and λ are the scattering angle and wavelength of the incident X-ray beam [1.70 Å], respectively) and the position of an incident X-ray beam on the detector were calibrated using several orders of layer reflections from silver behenate (d = 58.380 Å). Dynamic light scattering (DLS) measurements were performed with a Malvern model Zetasizer µV light scattering S5 spectrometer using an infrared laser (830 nm). Zeta potential measurements were performed using a Malvern model Zetasizer Nano ZSP zeta potential analyser. Confocal laser scanning microscopy (CLSM) was performed using a Leica model TCS SP8 microscope. Flow cytometry was performed using a BD model Accuri â C6 flow cytometer with BD Accuri C6 software in which the thresholds were set at 80,000 forward scatter (FSC) and at 0 side scatter (SSC) using detectors FL1 for green fluorescence and FL3 for red fluorescence to distinguish positive and negative sained cells . Asymmetric field flow fractionation was performed using an Eclipse model AF4 separation system equipped with a JASCO model UV-2070plus variable-wavelength UV-Vis detector. A regenerated cellulose membrane with a 5 kD cut-off (Wyatt Technology) and 350 µm spacer to adjust the channel thickness was used in combination with PIPES buffer (10 mM PIPES and 1 mM MgCl2, pH 6.8) as an eluent (detector-flow rate: 1.0 ml min -1 cross-flow rate: gradient from 0.5 ml min -1 to 0 ml min -1 for 180 min, transition time: 5 min). BSA was used as a standard for evaluating the concentrations of tubulin heterodimer (THD). Ultrafiltration was performed using cellulosemade centrifugal filters (Amicon ® Ultra-0.5 ml 100K) with a molecular weight cut-off of 100 kDa.
Concentrations of FITC molecules and gold nanoparticles were measured by determining the absorbance at 490 nm and 540 nm, respectively. All-atom molecular dynamics (MD) simulations S2 were performed in which all molecular systems were created using AmberTools 20 S3 and simulated with the GROMACS 2020.5 package S4 , and all images were created with the Visual Molecular Dynamics (VMD) package S5 .

1-3. Synthesis of compounds
Compound 2. To a dimethylformamide (DMF, 350 ml) solution of a mixture of 1 (15.9 g, 77.6 mmol) and Et3N (33.2 ml, 233 mmol) was added propargyl bromide (27.7 g, 233 mmol), and the mixture was stirred for 22 h at room temperature. Then, saturated aqueous NH4Cl (50 ml) was slowly added to the reaction mixture, and the resultant mixture was evaporated to dryness under reduced pressure. The residue was extracted with AcOEt (100 ml) and washed successively with saturated aqueous NH4Cl (50 ml × 2) followed by brine (50 ml). An organic extract separated was evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel with AcOEt/hexane (1/1 to 4/1) as an eluent to allow isolation of 2 as yellow oil in 54% yield (10.2 g).

S15
Compound 9: To a dry DMF (20 ml) solution of 2-(2-(2-chloroethoxy)ethoxy)ethanol (8, 2.5 g, 14.8 mmol) was added sodium azide (NaN3, 1.2 g, 17.8 mmol), and the mixture was stirred for 12 h at 65 °C. The reaction mixture was diluted with ether (50 ml) and filtered off from an insoluble fraction. The filtrate was washed with water (30 ml × 2) followed by brine (40 ml × 2). A separated organic extract was dried over Na2SO4 and filtered off from an insoluble fraction. The filtrate was evaporated to dryness under reduced pressure, and the residue was chromatographed on silica gel with AcOEt/hexane (1/1) as an eluent to allow for the isolation of 9 as a colorless oil (2.31 g, 89%).

S16
Compound 10: To a dichloromethane (CH2Cl2, 50 ml) solution of a mixture of 9 (2.0 g, 11.4 mmol) and Et3N (2.4 ml, 17.1 mmol) was dropwisely added p-toluenesulfonyl chloride (TsCl, 3.26 g, 17.1 mmol), and the mixture was stirred for 2 h at 0 °C and then for 2 h at room temperature. The reaction mixture was evaporated to dryness under reduced pressure, and the residue was diluted with CH2Cl2 (50 ml) and washed with hydrochloric acid (3%, 50 ml × 2), saturated aqueous NaHCO3 (50 ml × 2) and then brine (50 ml × 2). An organic extract separated was dried over Na2SO4 and filtered off from an insoluble fraction. The filtrate was evaporated to dryness under reduced pressure, affording 10 as a pale yellow oil (3.7 g, quant.).

Folding of Glue CO2in aqueous solution:
An atomistic molecular model of Glue CO2was created and parameterized according to the general AMBER force field (GAFF) S8 . As a first step, one Glue CO2molecule was placed in the center of a periodic simulation box filled with explicit TIP3P water molecules S9 . Neutralizing chloride (3) and sodium (1)
As a control, the [THDGTP*]3* system without Glue CO2was also simulated. All MD simulations have been conducted in explicit TIP3P water molecules S9 and in the presence of the necessary number of counterions to neutralize the systems.

Adhesions of Glue CO2on THDGTP*:
After preliminary minimization, all systems were first heated during two short MD runs under NVT (1 ns) and NPT (1 ns) periodic boundary conditions, during which the atoms of the THDGTP* were restrained (position restraint of 1000 kJ/mol/nm 2 ). During these phases, the system reached the simulation temperature of 37 °C, and the solvent density inside the simulation box was preadjusted.
After this preliminary equilibration, each system underwent a short MD simulation (20 ns) under NVT conditions at 37 °C while the THDGTP* was maintained at a fixed position to allow Glue CO2to approach the THDGTP* surface. Then, all restraints were removed, and all systems underwent 200 ns of MD simulation under NPT periodic boundary conditions at 37 °C and 1 atm. The AMBER99-ILDN force field was used to treat the protein topology S12 . A cut-off of 1.2 for both electrostatic and van der Waals interactions was used in the MD simulations. The Particle-Mesh Ewald method was applied to treat with a 1.2 nm cut-off for the Lennard-Jones interactions S13 . A v-rescale thermostat S14 with a coupling time step of 0.1 ps and Parrinello-Rahman barostat S15 with a reference pressure of 1 atm and coupling time step of 5.0 ps were used during the MD runs. The effects of Glue CO2on the hydrophobicity and total solvent-accessible surface area of the THDGTP* surface were calculated with the GROMACS gmx sasa tool S16 . Electrostatic potentials of the THDGTP* surface depending on the binding Glue CO2were studied using the Adaptive Poisson-Boltzmann Solver (APBS) software package S17 . All data have been extraced from the equilibrated phase MD trajectories.

Interactions of Glue CO2-:
To quantify the strength of the interactions of Glue CO2-, the radial distribution functions g(r) between key groups in the glues and in the tubulins were extracted from the MD trajectories. High and sharp peaks at short distances in g(r) identify a high relative probability to find groups close to S31 each other during the MD. This identifies the presence of strong and persistent interactions between groups, while no evident peaks and g(r) values <1 typically indicate no, or negligible, interactions (see Fig. 3j in the main manuscript). To assess the glue-tubulin interactions, g(r) curves were calculated between (i) the Gu + groups of Glue CO2and the anionic amino acids (aspartic acid and glutamic acid) of THDGTP*, (ii) the Gu + groups of Glue CO2and the OH groups of neutral amino acids (serine, threonine, and tyrosine) of THDGTP*, and (iii) the CO2groups of Glue CO2and the cationic amino acids (lysine and arginine) of THDGTP*. In addition, the g(r) were calculated to investigate the nature of Glue CO2--to-Glue CO2interactions. To this end, we estimated the g(r) for CO2vs. Gu + groups belonging to different Glue CO2molecules in the simulated systems.
Complete modeling data, structures and parameters used for, and extracted from simulations are available at https://zenodo.org/record/7070651#.Yx80t9JBxkg. Fig. 9. SAXS profile of NSGTP/GTP* (0.3 mg ml -1 ) in PIPES buffer (100 mM PIPES, 1 mM MgCl2, 250 µM GTP*, and 50 µM GTP, pH 6.8). The scattering intensity was proportional to q -2 in a small-q region S18 . Since two GTP molecules are hybridized to THDGTP, and one GTP and one GTP* molecule are hybridized to THDGTP*, the NMR results show that 65% of THDGTP* is contained in the NSGTP/GTP*.

2-1. Characterization of NSGTP/GTP* Supplementary
Signals marked with blue, magenta, orange, red, and green circles are assignable to protons in GTP and GTP*, which are highlighted in the corresponding colors.